Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A number of cell types possess an L-arginine-nitric oxide (NO) pathway. We studied the presence of constitutive and inducible forms of NO synthase in human platelets. N omega-nitro-L-arginine, an inhibitor of NO synthase, potentiated thrombin-induced aggregation of washed human platelets, whereas L-arginine inhibited it. The direct evidence for the presence of constitutive form of NO synthase came from the observation of conversion of tritium-labeled L-arginine to tritium-labeled L-citrulline by washed platelets suspended in Ca(++)-rich but not in Ca(++)-free buffer. Incubation of washed platelets in Ca(++)-free buffer with cytokines (tumor necrosis factor-alpha and interferon-gamma) or cytokines plus lipopolysaccharide caused a marked increase in the conversion of [3H]L-arginine to [3H]L-citrulline, suggesting the presence of inducible form of NO synthase. Gel electrophoresis identified an approximately 130 kd protein band with NO synthase in the platelet cytosol, which on isolation converted [3H]L-arginine to [3H]L-citrulline. This 130 kd protein required the presence of Ca++, reduced nicotinamide adenine dinucleotide phosphate tetrahydro-L-biopterin, and flavin adenine dinucleotide for expression of NO synthase activity. Platelet sonicates demonstrated presence of nitrite, and its concentrations were lowered by preincubation of platelets with NG-nitro-L-arginine methyl ester and enhanced in cytokine-treated platelets. Reverse-transcription polymerase chain reaction demonstrated messenger RNA expression of the constitutive endothelial (but not brain) and inducible isoforms of NO synthase in platelets. These observations indicate that human platelet cytosol possesses both constitutive and inducible forms of NO synthase.
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PMID:Identification of constitutive and inducible forms of nitric oxide synthase in human platelets. 753 7

Ovine tumour necrosis factor-alpha (OvTNF-alpha) was cloned by reverse transcription-polymerase reaction using RNA isolated from lipopolysaccharide (LPS)-stimulated alveolar macrophages and primers based on the human TNF-alpha cDNA sequence. An expression vector carrying the coding sequence of the mature form of ovine TNF was constructed. The recombinant Ov-TNF alpha (rOvTNF-alpha) was expressed as a glutathione-S-transferase (GST) fusion protein. It was cleaved with thrombin to yield rOvTNF free of the GST moiety. Growth at a lower temperature of 30 degrees C and use of Escherichia coli strains AM207, AM305, E392 and NM522 did not improve the recovery of rOvTNF-alpha from the soluble fraction to a significant extent. Purification of recombinant proteins was achieved rapidly and easily by affinity chromatography using glutathione-Sepharose. Yields of pure rOvTNF-alpha achieved in E. coli JM109 and AM207 were approximately 1 mg L-1. Both rOvTNF-alpha and recombinant human TNF-alpha (rhTNF-alpha) exerted cytotoxicity on L929 cells. However, rOvTNF-alpha but not rhTNF-alpha stimulated proliferation of ovine thymocytes. Maximum levels of TNF-alpha mRNA expression by LPS-stimulated ovine alveolar macrophages were detected at approximately 4 h post-stimulation.
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PMID:Expression, biological activity and kinetics of production of recombinant ovine TNF-alpha. 753 48

We compared peritoneal dialysis effluents from 18 CAPD patients who had not suffered from peritonitis during the last 6 months (group 1) with the effluents from five patients with acute peritonitis (group 2), measuring activation markers of coagulation and fibrinolysis. These markers included prothrombin fragment F1 + 2 (F1 + 2), thrombin-antithrombin III complex (TAT), fibrin monomer (FM), and fibrin degradation products (FbDP). In the dialysate of group 1 we found remarkably high levels of F1 + 2, TAT and FM concomitant with a high concentration of FbDP, indicating a high rate of intraperitoneal fibrin turnover. The balance between peritoneal generation and degradation of fibrin was disturbed in untreated patients of group 2, who had significantly higher levels of coagulation markers and a higher ratio between FM and FbDP. Seven days after treatment with intraperitoneal administration of antibiotics and heparin, F1 + 2, TAT, FM and FbDP decreased significantly. To evaluate the role of mesothelial cells (MC) in the high peritoneal fibrin turnover we investigated the expression of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor type-1 (PAI-1), and tissue factor in cultured human peritoneal MC under basal conditions and after exposure to tumour necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha), or bacterial lipopolysaccharide (LPS). The exposure of MC to TNF alpha or to a lesser extent IL-1 alpha or LPS reduced their fibrinolytic activity by decreasing t-PA production and increasing PAI-1 synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Imbalance between intraperitoneal coagulation and fibrinolysis during peritonitis of CAPD patients: the role of mesothelial cells. 756 82

Fibrin deposition is characteristic of inflammatory diseases. The monocytes is central to the inflammatory response and can affect fibrinolysis by expression of urokinase (u-PA) and plasminogen activator inhibitor types 1 and 2 (PAI-1 and PAI-2, respectively). This study examines whether thrombin, which promotes fibrin deposition, can contribute to fibrin persistence by modulating expression of proteins of the fibrinolytic system. Monocytes were isolated from human peripheral blood and analyzed for PAI-2, PAI-1, and u-PA antigens by enzyme-linked immunosorbent assay (ELISA). Monocytes responded to thrombin by increased expression of PAI-2 in a dose- and time-dependent manner, with maximal synthesis at a concentration of 1 U/mL to 10 U/mL. This trend was also evident for PAI-1, which was present at much lower levels. Thrombin and lipopolysaccharide (LPS) stimulated comparable levels of PAI-2, studied at the antigen and mRNA level. The dose effet of LPS on PAI-2 and PAI-1 was found to differ from that of thrombin. The level of u-PA was undetectable by ELISA and zymography in all samples. Thrombin stimulates PAI-2 synthesis by human monocytes, therefore creating an imbalance in the fibrinolytic system. This may contribute to persistence of fibrin, deposited during inflammation.
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PMID:Thrombin modulates synthesis of plasminogen activator inhibitor type 2 by human peripheral blood monocytes. 757 47

Circulating monocytes and vascular endothelial cells (EC) interact in a complex and dynamic manner that varies between vascular beds. The objective of this study was twofold: to ascertain if monocytic cell adhesion to vascular endothelium differed between specific anatomic regions of the canine aorta, and to investigate the effect of known EC stimulators on monocytic cell adhesion to cells from these regions. Initial in vitro studies measuring adherence of U937 cells, a human monocytic cell line, to canine jugular vein and aortic EC monolayers revealed a dose-dependent increase in adhesion to EC stimulated with interleukin-1 (IL-1), lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), or thrombin. While there was no regional difference in monocytic cell adherence to unstimulated EC in tissue culture, studies demonstrated greater monocytic cell adhesion to stimulated EC cultured from the distal versus proximal aorta. In organ culture, unstimulated adhesion of U937 cells or autologous monocytes was significantly greater to the distal aorta than the proximal aorta. Although monocytic cell adhesion to both the proximal and distal aorta increased with stimulation, the percentage increase in the proximal aorta, 1,086% with IL-1, 237% with PMA, 209% with LPS, and 174% with thrombin, was greater than in the distal aorta, demonstrating a significant functional difference in the endothelium from separate anatomic regions of a single vessel. This may have a direct relevance to the regional specificity of vascular disease.
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PMID:Differential monocytic cell adherence to specific anatomic regions of the canine aorta. 765 83

We investigated the responses of canine coronary rings to endothelium-derived relaxing factor-nitric oxide- (EDRF-NO) dependent agonists and NO synthase (NOS) inhibitors 3 h after endotoxic shock was induced in dogs by lipopolysaccharide infusion (LPS; 2 mg/kg). EDRF-NO-dependent relaxation to thrombin [control maximum response produced after administration of thrombin (Emax) was -85.2 +/- 7.0% of the constrictor response produced by the thromboxane analogue U-46619], acetylcholine (control Emax -88.4 +/- 3.4%), or bradykinin (control Emax -80.5 +/- 2.2%) was not inhibited by LPS (Emax thrombin -75.9 +/- 9.5%; Emax acetylcholine -90.2 +/- 2.4%; Emax bradykinin -91.6 +/- 3.4%). The NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) (10(-6)-3 x 10(-4) M) caused constriction of rings with endothelium (Emax 36.3 +/- 5.6%), an effect that was greater after LPS (Emax 59.2 +/- 4.1%; P < 0.05). D-NMMA had no effect in control, but it increased tension after LPS (Emax 20.8 +/- 9.7%). Contrary to expectations, L- and D-NMMA relaxed endothelium-denuded rings (-30.4 +/- 8.7% L-NMMA; -45.1 +/- 11.7% D-NMMA; P < 0.05). However, neither agent caused relaxation after in vivo LPS (10.2 +/- 3.4% L-NMMA; 8.9 +/- 5.2% D-NMMA). N omega-nitro-L-arginine-methylester (L-NAME) and nitro-L-arginine (10(-6)-3 x 10(-4) M) increased tension (Emax 82.3 +/- 23.9 and 73.1 +/- 8.8%, respectively) but only when endothelium was present, and the increases were no greater in LPS-treated groups than in controls (with LPS: Emax L-NAME 87.3 +/- 16.5%; Emax nitro-L-arginine 65.7 +/- 3.3%).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of NG-substituted arginines on coronary vascular function after endotoxin. 769 Jul 46

1. Hypotension and vascular hyporesponsiveness to vasoconstrictors are observed during endotoxic shock, and are associated with increased production of nitric oxide in the vascular wall. Disseminated intravascular coagulation is another feature of septicaemia. We hypothesized that thrombin generated during disseminated intravascular coagulation might modulate the changes in vascular tone induced by endotoxin. 2. Incubation of rat aortic rings for 4 h with alpha-thrombin (0.003-3.0 NIH units/ml) did not change their reactivity to noradrenaline. Incubation for 4 h with lipopolysaccharide increased the EC50 for noradrenaline, whereas co-incubation of thrombin (0.5 NIH units/ml) with lipopolysaccharide did not alter this hyporeactivity to noradrenaline. 3. In vivo in rats, lipopolysaccharide caused early (1 h) and late (4-6 h) hyporeactivity to noradrenaline. In rats infused with lipopolysaccharide and heparin (1 U min-1 kg-1, 0.4 ml/h) or hirudin (2.2 mg ml-1 kg-1, 0.8 ml/h), vasopressor responses to noradrenaline were not different from those after infusion of lipopolysaccharide alone. Aortic rings taken from rats receiving both anticoagulant treatment and lipopolysaccharide had the same sensitivity to noradrenaline as those obtained from rats receiving lipopolysaccharide alone. 4. Our results suggest that, in vivo, disseminated intravascular coagulation does not modify the early and late effects of lipopolysaccharide on arterial pressure and that, in vitro, thrombin neither induces hyporeactivity to noradrenaline nor modifies lipopolysaccharide-induced hyporeactivity. We propose that thrombin generated during disseminated intravascular coagulation in rats does not play a major role in the alterations of vascular tone observed during endotoxic shock.
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PMID:Thrombin does not alter vascular hyporeactivity in models of endotoxin-induced septic shock in rats. 772 Mar 38

Endothelin-1 (ET-1) is known to be involved in a variety of pathophysiologic conditions, especially of the pulmonary vasculature. The aim of this study was to investigate physiologic mediators potentially involved in the pathogenesis of pulmonary hypertension, for their effects on ET-1 gene expression at both the transcriptional and translational level. Rat microvascular and pulmonary artery endothelial cells grown in culture were exposed to vasoactive mediators (thrombin or an anoxic gas mixture) and inflammatory mediators (lipopolysaccharide, interleukin 1 alpha, interleukin 1 beta, or tumor necrosis factor alpha) for various time periods. The change in prepro-ET-1 (ppET-1) mRNA levels in these cells in response to stimuli was a time-dependent phenomenon. The inflammatory mediators caused an acute rise in ppET-1 mRNA levels whereby peak induction occurred after 1 h with a rapid decline to control levels by 4 h. The vasoactive mediators elicited a more sustained response whereby a significant elevation in ppET-1 mRNA expression occurred quickly and remained elevated through 4 h. The pattern of induction was more rapid for thrombin than for anoxic gas exposure. Radioimmunoassay analysis demonstrated a similar response for thrombin and the inflammatory mediators in ET-1 mature peptide release, whereas the effect of anoxic gas exposure was divergent. Significant elevations were noted after 6 h for thrombin as well as each of the inflammatory mediators except IL-1 alpha. In response to the anoxic gas exposure, however, a significant rise in ET-1 peptide release was not evident until after 24 h. To determine the level at which ppET-1 mRNA induction is regulated, cells were cotreated with each of the stimuli and actinomycin D or cycloheximide. Results indicate that the induction of ppET-1 mRNA levels is likely due to de novo transcription, as well as mRNA stabilization. In summary, inflammatory and vasoactive agents are important regulators of ET-1 gene expression in rat pulmonary endothelial cells; most important, we observed a differential response at the mRNA or peptide level depending on the mediator involved.
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PMID:Effects of vasoactive and inflammatory mediators on endothelin-1 expression in pulmonary endothelial cells. 774 14

Lactoferrin is a prominent component of neutrophil secondary granules, and its blood concentration is increased in certain inflammatory diseases. In contrast to the well-described biochemical characterization of lactoferrin as an iron-binding protein, its physiologic role in the regulation of inflammation and other host defense mechanisms is unclear. In this report, we provide evidence that lactoferrin has a potent heparin-neutralizing activity during thrombin inhibition by the serine proteinase inhibitors (serpins) antithrombin and heparin co-factor II. Activated neutrophil supernatant, which contains lactoferrin and other heparin-binding proteins, could neutralize the heparin-dependent antithrombin-thrombin inhibition reaction. The addition of lactoferrin to plasma corrected the heparin-induced prolongation of blood plasma coagulation as measured by the activated partial thromboplastin time (aPTT). Treatment of whole blood with specific inflammatory mediators, fMLP, lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) increased the concentration of both plasma lactoferrin and platelet factor 4 while inhibiting the blood anticoagulant activity of heparin as measured by the aPTT. These results suggest that the prothrombotic sequelae of some inflammatory processes may be partly due to various agonists that release neutrophil lactoferrin, which can then neutralize glycosaminoglycan-dependent serpin-thrombin inhibition reactions.
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PMID:Neutralization of heparin activity by neutrophil lactoferrin. 781 95

Endothelial damage, synovial oedema, fibrin deposition, polymorphonuclear cell (PMN) invasion, and mild lining cell hyperplasia characterize acute inflammatory arthritis. Later on, perivascular tissue is infiltrated by mononuclear cells. The early events are mediated by interactions between PMNs and endothelial cells. Both parts in the adhesion event are activated with multiple stimuli resulting in complex interactions of varying intensity and duration. Adhesion molecules present on the surface of PMNs (L-selectin) or induced by inflammatory stimuli (beta 2-integrins) mediate PMN adhesion to activated endothelium, which has counter receptors (E-selectin for L-selectin and ICAM-1 and ICAM-2 for beta 2-integrins). At the initial phase L-selectin initiates the rolling of PMNs on endothelial cells. Further stimuli result in a more prolonged adhesion between PMNs and endothelium. At the side of endothelium, induction of P-selectin and PAF by histamine, thrombin and LTC4 contribute to the acute rolling of PMNs on endothelial surface. Tumor necrosis factor (TNF), interleukin-1 (IL-1) and lipopolysaccharide activate endothelial cells to synthesize interleukin-8 (IL-8), a potent chemotactic and proadhesive mediator for PMNs, and further adhesion molecule (E-selectin), a mediator of long-term adhesion between PMN and endothelium. After adhesion and migration to the focus of inflammation, PMNs induce inflammation by aggregating, releasing hydrolyzing enzymes, generating lipid peroxidation products such as prostaglandins and LTB4, and oxygen derived free radicals. In studies on the pathogenesis of seronegative spondyloarthropathies, we have shown persistently aberrant PMN function evidenced by enhanced chemotaxis and high production of toxic oxygen derived free radicals by PMN.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The present knowledge of the inflammatory process and the inflammatory mediators. 781 74


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